Chromatography is a set of techniques for separating a mixture into its constituents. In liquid chromatography systems, generally, one or more high-pressure pumps take in solvents and deliver a liquid solvent composition to a sample manager, where a sample awaits injection into the mixture. The sample is the material under analysis, examples of which include complex mixtures of proteins, protein precursors, protein fragments, reaction products, and other compounds, to list but a few. From the sample manager, the resulting liquid composition, comprised of the mixture of solvents and injected sample, moves to a point of use, such as a column of particulate matter. By passing the composition through the column, the various constituents in the sample separate from each other at different rates and thus elute from the column at different times. A detector receives the elution from the column and produces an output from which the identity and quantity of the analytes may be determined.
High-performance liquid chromatography (HPLC) uses two basic elution modes: isocratic elution and gradient elution. In the isocratic elution mode, the mobile phase, comprised of either a pure solvent or a mixture of solvents, remains the same throughout the chromatography run. In the gradient elution mode, the composition of the mobile phase changes during the separation. Creation of the gradient (i.e., changing mobile phase composition) entails the mixing of multiple solvents, the proportions of which change over time in accordance with a predetermined timetable. Some HPLC systems create the gradient under high pressure, by mixing the solvents downstream of the pumps. Such HPLC systems are also referred to herein as high-pressure gradient systems. Other HPLC systems create the gradient under low pressure, using a gradient proportioning valve to select from up to four solvents, mixing the multiple solvents in front of a single aspirating pump, and changing the proportions of the solvents over time. Such HPLC systems are also referred to herein as low-pressure gradient systems.
The decision between a high-pressure and a low-pressure gradient system involves a variety of tradeoffs, only a few of which are mentioned here. For one, high-pressure gradient systems have lesser dwell volumes than low-pressure gradient systems because the solvent mixing occurs after the pumps instead of before the intake side of the pump. However, because of the location of mixing, low-pressure gradient systems can produce a gradient with just one pump, whereas high-pressure gradient systems generally require one pump for each solvent. Hence, low-pressure gradient systems are more amenable than high-pressure gradient systems to tertiary and quaternary gradients, and are thus predominantly used for such chromatography applications, whereas high-pressure gradient systems are generally used for binary gradients.
Often, however, it is desirable to blend more than two solvents in a gradient, with a third solvent being a modifier, such as TFA (triflouroacetic acid), introduced at a constant percentage. Furthermore, it is easier to blend in a more concentrated mixture of the modifier to the total composition than to add the modifier to each of the other solvents at the desired lower concentration. For example, if 0.1% TFA is the desired concentration, it is much easier to produce a 1% concentration of TFA, and introduce it in 10% proportion to the other two solvents, than to mix a 0.1% concentration of TFA in each of the other two solvents. Hence, a low-pressure gradient system is generally used for chromatography runs to introduce the third modifier solvent instead of a high-pressure gradient system. Use of the low-pressure gradient system for this purpose, though, has disadvantages of an increased dwell volume (in comparison to a high-pressure gradient system) and limiting the maximum percentage of one of the two other solvents to 90%.